This study was supported by Fondi Ricerca di Ateneo (FRA)2015-2016, University of Sannio.

1. Immunoperoxidase protocol
NOTE: The zebrafish were obtained by Prof. Omid Safari (Department of Fisheries, Faculty of Natural Resources and Environment, Ferdowsi University of Mashhad, Mashhad, Iran)10.
<ol>
	<li>Tissue dissection
	<ol>
		<li>Sacrifice the zebrafish by submersion in ice water (5 parts ice/1 part water, 4 &#176;C); leave them until cessation of all movement to ensure death by hypoxia.</li>
		<li>Quickly remove the gut and brain with...

The authors declare no conflicts of interest.

Sample preparation
Sample preparation is the first critical step in IHC. A reliable protocol allows for maintenance of cell morphology, tissue architecture, and antigenicity. This step requires correct tissue collection, fixation, and sectioning22,23. The purpose of fixation is to preserve tissue and reduce the action of tissue enzymes or microorganisms. In particular, the fixation step preserves cellular compon...

Immunohistochemistry (IHC) is a well-established classic technique used to identify cellular or tissue components (antigens) by antigen-antibody interaction1,2. It can be used to identify the localization and distribution of target biomolecules within a tissue. IHC uses immunological and chemical reactions to detect antigens in tissue sections3. The main markers used for the visualization of antigen-antibody interactions include fluorescent dyes (immunofluorescence) and enzyme-substrate color reactions (immunoperoxidase), both conjugated to antibodies4. Using microscopic observation is possible to determine the localization of labeled tissue, which approximately corresponds to localization of the target antigen in the tissue.
Two methods exist for fluorescent or chromogenic reactions to detect protein: the direct detection method, in which the specific primary antibody is directly labeled; and the indirect detection method, in which the primary antibody is unconjugated while the secondary antibody carries the label5,6,7. The indirect method has some advantages, which is mainly its signal amplification. Moreover, unlike other molecular and cellular techniques, with immunofluorescence, it is possible to visualize the distribution, localization, and coexpression of two or more proteins differentially expressed within cells and tissues7. The choice of the detection method used depends on experimental details.
To date, IHC is widely used in basic research as a powerful and essential tool to understanding the distribution and localization of biomarkers and the general profiling of different proteins in biological tissue from human to invertebrates8,9,10,11. The technique helps display a map of protein expression in a large number of normal and altered animal organs and different tissue types, showing possible down- or up-regulation of expression induced by physiological and pathological changes. IHC is a highly sensitive technique that requires accuracy and the correct choice of methods to obtain optimal results12. First of all, many different factors such as fixation, cross-reactivity, antigen retrieval, and sensitivity of antibodies can lead to false positive and false negative signals13. Selection of the antibodies is one of the most important steps in IHC and depends on the antigen specificity and its affinity to the protein and species under investigation7.
Recently, we have optimized the IHC technique to detect members of orexin/hypocretin and endocannabinoid systems in adult zebrafish tissue. We have focused mainly on fixation, tissue embedding using two different approaches, sectioning and mounting (which can affect resolution and detail during microscopic analysis), and blocking (to prevent false positives and reduce background)14. Other important characteristics are the antibody specificity and selectivity and reproducibility of individual IHC protocols. The key to providing antibody specificity is the use of negative controls (including no primary antibodies or tissue that is known to not express the target proteins) as well as positive controls (including tissue that is known to express the target proteins)15. The selection of antibodies for IHC is made based on their species-specificity (the likelihood with which they react with the antigen of interest) and the antigen-antibody binding detection systems that is used4,5,6,7. In the case of immunoperoxidase, the color of the reaction is determined by selection of the precipitating chromogen, usually diaminobenzidine (brown)16. On the other hand, immunofluorescence utilizes antibodies conjugated with a fluorophor to visualize protein expression in frozen tissue sections and allows for easy analysis of multiple proteins with respect to the chromogenic detection system5,7.
In the immunoperoxidase technique, the secondary antibody is conjugated to biotin, a linker molecule capable of recruiting a chromogenic reporter molecule [avidin-biotin complex (ABC)], leading to amplification of the staining signal. With the ABC reporter method, the enzyme peroxidase reacts with 3,3&#8217;-diaminobenzidine (DAB), producing an intensely brown-colored staining where the enzyme binds to the secondary antibody, which can then be analyzed with an ordinary light microscope. ABC staining, due to the high affinity of avidin for biotin, produces a rapid and optimal reaction, with few secondary antibodies attached to the site of the primary antibody reactivity. This chromogenic detection method allows for the densitometric analysis of the signal, providing semi-quantitative data based on the correlation of brown signal levels with protein expression levels18.
With immunofluorescence techniques, simultaneous detection of multiple proteins is possible due to the ability of different fluorochromes to emit light at unique wavelengths, but is important to choose fluorochromes carefully to minimize spectral overlap5. Moreover, the use of primary antibodies in different host species minimizes difficulties concerning cross-reactivity. In this case, each species-specific secondary antibody recognizes only one type of primary antibody. Fluorescent reporters are small organic molecules, including commercial derivatives, such as Alexa Fluor dyes.
Many animal models are used to understand particular physiological and pathological conditions. To date, it is established that many metabolic pathways are conserved over the course of evolution. Therefore, IHC studies in model organisms such as zebrafish can provide insight into the genesis and maintenance of pathological and non-pathological conditions17. It is an aim of this report to illustrate IHC protocols that can be performed on adult zebrafish tissue and used to obtain detailed images of the distribution and localization of OXA, OX-2R, and CB1R at peripheral and central levels. Also reported are protocols for the application of two major IHC indirect methods in peripheral and central tissues of adult zebrafish. Described is the indirect method, which allows for signal amplification in cases where a secondary antibody is conjugated to a fluorescent dye (immunofluorescence method) or enzyme reporter (immunoperoxidase method). Both chromogenic and fluorescent detection methods possess advantages and disadvantages. Reported in this protocol is the use of IHC, mainly immunofluorescence, in adult zebrafish, an animal model widely used to study systems that are evolutionary conserved across different physiological and pathological conditions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti CB1</td>
			<td>Abcam</td>
			<td>ab23703</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti OX-2R</td>
			<td>Santa Cruz</td>
			<td>sc-8074</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-OXA</td>
			<td>Santa Cruz</td>
			<td>sc8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Aquatex</td>
			<td>Merck</td>
			<td>1,085,620,050</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated rabbit anti-goat</td>
			<td>Vector Lab</td>
			<td>BA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon</td>
			<td>Nikon Eclipse Ti2</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystem</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>32670</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera</td>
			<td>Leica Biosystem</td>
			<td>DFC320</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera for confocal microscope</td>
			<td>Nikon</td>
			<td>DS-Qi2</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti goat Alexa fluor 488-conjugated secondary antibodies</td>
			<td>Thermo Fisher</td>
			<td>A11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti goat Alexa fluor 594-conjugated secondary antibodies</td>
			<td>Thermo Fisher</td>
			<td>A11058</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti rabbit Alexa fluor 488-conjugated secondary antibodies</td>
			<td>Thermo Fisher</td>
			<td>A21206</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti rabbit Alexa fluor 594-conjugated secondary antibodies</td>
			<td>Thermo Fisher</td>
			<td>A21207</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td>20,821,330</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen section compound</td>
			<td>Leica Biosystem</td>
			<td>FSC 22 Frozen Section Media</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O2</td>
			<td>Sigma Aldrich</td>
			<td>31642</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>VWR</td>
			<td>20,252,290</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPACT DAB</td>
			<td>Vector lab</td>
			<td>SK4105</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Biosystem</td>
			<td>DMI6000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystem</td>
			<td>RM2125RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma Aldrich</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4H2O</td>
			<td>Sigma Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Sigma Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Rabbit Serum</td>
			<td>Vector lab</td>
			<td>S-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin wax</td>
			<td>Carlo Erba</td>
			<td>46793801</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraphormaldeyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium citrate dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>W302600</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka Analytical</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma</td>
			<td>Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaStain Elite ABC Kit</td>
			<td>Vector lab</td>
			<td>PK6100</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene Pure</td>
			<td>Carlo Erba</td>
			<td>392603</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of orexin and endocannabinoid receptors in adult zebrafish using immunoperoxidase and immunofluorescence methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Representative data for the immunoperoxidase staining are shown in Figure 1 and Figure 2. Immunohistochemical analysis of OX-A and OX-2R distribution in the gut of adult zebrafish showed different localization sites of OX-A and OX-2R and their increases in expression in the intestinal cells of DIO zebrafish. An intense brown staining for OX-A was observed in the cells of the medial and anterior intestine (Figure 1A, A1). ...

Watch this Scientific Journal Video about Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods at JoVE.com

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

Currently, immunohistochemistry is used with increasing frequency to identify the presence of specific molecular markers and their changes in case of pathologies, giving root to the possibility to perform quantitative analysis. Immunohistochemistry, mainly the immunofluorescence technique, is used in the larval and the adult zebrafish animal model, but little is known about a good standard immunohistochemistry protocol for zebrafish. Here, we describe and illustrate a protocol that can be used to study the evolutionary conservation of important proteins in adult zebrafish.A good protocol for immunohistochemistry in adult zebrafish can help to underlie the usefulness of this animal model to study the morphological expression and distribution of highly conserved biomolecules. This method can also help to analyze possible alterations of these biomolecules due to pathological conditions correlated to human physiopathology. Demonstrating the procedure will be Dr.Lea Tunisi, a PhD student from my laboratory.To begin this procedure, transfer the prepared frozen tissue blocks to a cryostat at minus 20 degrees Celsius. Cut the block in coronal or sagittal sections of 10 micrometers. Then, collect the tissues in alternate serial sections onto adhesive glass slides suitable for immunohistochemistry, and store them at minus 20 degrees Celsius until ready to proceed.First, use a solvent-resistant pen to demarcate the tissue area on the slide. Rinse the sections three times for five minutes each using 0.1-molar phosphate buffer. Next, dissolve 0.3 milliliters of Triton X-100 in 100 milliliters of 0.1-molar phosphate buffer to prepare Triton X-100 0.3%Dissolve 0.1%normal donkey serum in phosphate buffer containing 0.3%Triton X-100 to prepare the blocking solution.Incubate the sections with the solution of normal donkey serum dissolved in permeabilization buffer PB-Triton X-100 0.3%for 30 minutes at room temperature to permeabilize the cell membrane and block the nonspecific binding sites. First, rinse the sections three times for five minutes each with 0.1-molar phosphate buffer. Add the mix of primary antibodies diluted in PB-T, and incubate the sections overnight in a humid box at room temperature.The day after, rinse the sections three times for five minutes each with 0.1-molar phosphate buffer. Incubate sections at room temperature for two hours in an appropriate mix of secondary antibodies diluted in PB-T. To begin, rinse the sections three times for five minutes each with 0.1-molar phosphate buffer.Dissolve 1.5 microliters of DAPI in three milliliters of phosphate buffer to prepare the nuclear dye DAPI. Counterstain the sections in the dye. Then, use mounting medium to coverslip the slides to stabilize the tissue and stain for long-term usage.Fluorescent samples can be stored in the dark at four degrees Celsius. Repeat the immunofluorescence protocol, and either omit the primary or secondary antibody, or substitute the primary or secondary antiserum with phosphate buffer to prepare the negative control. Next, repeat the immunofluorescence protocol, and pre-absorb each primary antibody with an excess of the relative peptide.Repeat the immunofluorescence protocol on slices of mouse brain to prepare the positive control. Using a confocal microscope equipped with an X-Y-Z motorized stage, a digital camera, and acquisition and analysis software, observe and analyze the immunostained sections. Acquire digital images with the 5x, 20x, and 40x objectives.Take images of each section at low magnification in each of the available channels to compose a low-magnification montage. Normalize the fluorescence images to maximum contrast and overlay. Throughout the area of interest, collect serial Z-stacks of images through six focal planes with focal steps of one to 1.8 micrometers.Perform this collection for each channel separately. After this, use imaging deconvolution software to deconvolve images by application of 10 iterations, and collapse the serial Z-plane images into a single maximum projection image. Use an appropriate image editing software to adjust the micrographs for light and contrast.Immunohistochemical analysis of OX-A and OX-2R distribution in the gut of adult zebrafish shows different localization sites of OX-A and OX-2R and their increases in expression in the intestinal cells of DIO zebrafish. An intense brown staining for OX-A is observed in the cells of the medial and anterior intestine. The immunoexpression of OX-A gives clear signals in the different gut compartments, decreasing from the anterior toward the medial intestine.Similar results are observed for OX-2R immunoexpression in the intestine of DIO and control diet zebrafish. An increased OX-A signal in DIO adult zebrafish is accompanied by the overexpression of OX-2R in other intestinal compartments. Accurate analysis of immunofluorescent images shows the increase of OX-2R/CB1R colocalization in the gut of DIO adult zebrafish compared to the control diet zebrafish.A similar situation is observed in different brain regions, such as the dorsal telencephalon, hypothalamus, optic tectum, torus lateralis, and diffuse nucleus of the inferior lobe. By double immunostaining with orexin-A and CB1R colocalization, it is observed that there was an increase of colocalization in the orexinergic neurons of the hypothalamus, accompanied by an increase of orexin-A fluorescent signal. These results show how double immunofluorescence can help to identify physiologically conserved protein expression, colocalization of target proteins, and their distribution and/or expression changes in different pathological conditions.Using immunofluorescence, we can better understand the codistribution and coexpression of biomolecules, their possible interactions, and we can also have a visible image of their changes in case of different pathologies. Moreover, the neuroanatomical distribution of metabolic enzyme expression or receptors may further reveal the role of protein signaling within tissues. The present technical work introduced the immunofluorescence approach to study two highly conserved system, orexin and endocannabinoids, in using an adult zebrafish model.Using this protocol described here for the first time in the brain of adult zebrafish, the anatomical distribution and coexpression of orexin-2 receptors and the endocannabinoid-CB1 receptors hasn't been determined. We recommend the protocols described here for immunohistochemistry experiments and to detect spatial distribution and organization of receptors in peptides in adult zebrafish to better understand their functions.

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Research

JoVE Journal

Biochemistry

7.9K visualizações.  University of Sannio. Atualmente, a imunohistoquímica é utilizada com frequência crescente para identificar a presença de marcadores moleculares específicos e suas alterações no caso de patologias, dando raiz à possibilidade de realização de análise quantitativa. A imunohistoquímica, principalmente a técnica de imunofluorescência, é usada no modelo animal larval e zebrafish adulto, mas pouco se sabe sobre um bom protocolo imunohistoquímico padrão para zebrafish. Aqui, descrevemos e ilustramos um p...